Co-translational interactions play a crucial role in nascent chain modifications targeting folding and assembly pathways. Here, we describe selective ribosome profiling, a method for in vivo direct analysis of these interactions in the model eukaryote Saccaromyces cerevisiae. Selective ribosome profiling is the only method to date that captures and characterizes co-translational interactions in vivo in a direct manner.
Selective ribosome profiling enables global profiling of any factors and interactions with translating ribosomes in near-codon resolution. Begin by collecting the constructed yeast cells containing the desired tagged proteins. Perform cell lysis using cryogenic grinding in a mixer mill twice for two minutes at 30 Hertz.
Centrifuge for two minutes at 30, 000 times G at four degrees Celsius to clear the lysate, and collect the supernatant. Transfer 200 microliters of the supernatant into a microcentrifuge tube for total RNA analysis and 700 microliters into a microcentrifuge tube for immuno-purification. Digest the previously separated total RNA sample using 10 units of RNase I for 25 minutes at four degrees Celsius on a rotating mixing rack at 30 rotations per minute.
Prepare the sucrose cushion master mix as described in the text manuscript. Then, load the sample onto 400 microliters of the sucrose cushion and centrifuge for 90 minutes at 245, 000 times G and at four degrees Celsius. Remove the supernatant quickly with a vacuum pump and overlay pellets with 150 microliters of lysis buffer.
Secure the sample with paraffin wrap and resuspend the pellets by shaking for one hour at four degrees Celsius at 300 rotations per minute. Wash 100 to 400 microliters of the affinity binding matrix per sample three times with one milliliter of lysis buffer by resuspending the affinity matrix in the lysis buffer. Then, rotate at 30 rotations per minute with a rotating mixing rack at four degrees Celsius for five minutes.
Precipitate by centrifugation. Discard the upper liquid and repeat this process three times. Add affinity binding matrix to the previously separated immuno-purification sample and digest it using RNase I.Rotate for 25 minutes at 30 rotations per minute and four degrees Celsius with a rotating mixing rack to bind the protein to the affinity matrix.
Prepare the wash buffer master mix as described in the text manuscript. Wash the affinity binding matrix with one milliliter of wash buffer for approximately one minute, rotating in the mix rack. Next, precipitate by centrifugation at 3000 times G for 30 seconds at four degrees Celsius and discard the upper liquid.
Repeat three times. Wash twice more in one milliliter of wash buffer, each time for five minutes, rotating in the mix rack. Precipitate again by centrifugation.
Use 50 microliters of beads for protein elution with the same amount of 2X sample buffer. Centrifuge to pellet the beads and discard the upper liquid. Elute with 700 microliters of 10-millimolar Tris.
Then, freeze in liquid nitrogen. Remove the sample from liquid nitrogen and store at minus 80 degrees Celsius to be used for subsequent RNA extraction. Assess the success of the affinity purification step by Western blot or Coomassie staining with aliquots of each step using mock IP on a non-tagged, wild-type strain as a control for non-specific binding to the affinity matrix.
Western blot results after affinity purification demonstrated the success of the tagged protein expression. Isolation of ribosome-protected footprints was validated using the results from the bioanalyzer. While generating a cDNA library for deep sequencing and big data analysis, under-cycling leads to low yield.
However, with the dNTPs still present, the reaction proceeds, generating longer PCR artifacts with chimeric sequences due to PCR products priming themselves. The generated library was further validated by high-sensitivity DNA electrophoresis for exact size distribution and quantification. The sequence library was trimmed for adapters and barcodes and the resulting reads were divided into different groups of coding sequences, introns, and intergenic sequences.
Ribosome profiles analyzing co-translational interactions of Vma2p with ribosome-associated chaperone Ssb1p, Pfk2p, and Fas1p are shown here with the experimental scheme of each affinity purification. There was ribosome-occupancy along the open reading frame of total translatomes compared to Ssb1, Pfk2p, and Fas1p interactomes. Mean enrichment of Ssb1p, Pfk2p, and Fas1p at each ribosome position along the open reading frame is shown here.
This method enables identification and characterization of the various co-translational interactions, ensuring a functional proteome, as well as the study of various diseases. To date, selective ribosome profiling is the only method that can capture and characterize these interactions in a direct manner in vivo.
